Mononuclear non-heme-iron (MNH-Fe) enzymes activate O2 for a stunning array of biomedically, agriculturally, and environmentally important oxidation reactions. Our past decade's work, supported (in part) by this grant, established the intermediacy of iron(IV)-oxo (ferryl) complexes in the reactions of seven different MNH-Fe enzymes. Five of these complexes generate substrate radicals by abstracting hydrogen (H) from unactivated aliphatic carbons, initiating formation of new C-O, C- Cl/Br, and C-S bonds. Energized by our recent success in rationalizing the divergent outcomes mediated by the (halo)ferryl complexes in the -ketoglutarate(KG)-dependent aliphatic hydroxylases and halogenases, we now aim to understand even more complex ferryl-mediated transformations, including those exhibited by the enzymes: (1) hydroxypropylphosponate epoxidase (HppE), which catalyzes the 1,3-dehydrogenation of an alcohol to an epoxide, using hydrogen peroxide as the oxidant, in the biosynthesis of the antibiotic, fosfomycin; (2) carbapenem synthase (CarC), which uses one or more tyrosyl radical in concert with the presumptive ferryl complex to promote stereoinversion of a chiral carbon and desaturation of a C-C bond two atoms removed from the stereocenter, reportedly in a single O2 activation event, to produce the core of an important class of antibiotics; and (3) 2-hydroxyethylphosponate (2-HEP) dioxygenase (HEPD) and methylphosphonate synthase (MPnS), a pair of related enzymes that use ferryl complexes to cleave the C-C bond of 2- HEP in distinct 4-e- oxidation reactions, producing a precursor to the herbicide phosphinothricin (HEPD) and a major store of oceanic methane (MPnS). Our past studies on myo-inositol oxygenase and isopenicillin N synthase demonstrated a fundamentally distinct manifold for enzymatic O2 and C- H activation, involving H abstracting FeIII-superoxo complexes. This manifold obviates the requirement for a reducing co-substrate (e.g., KG), enabling four-electron (4-e-) oxidations. HEPD and MPnS are likely also to employ this manifold on the pathways to their ferryl intermediates, a hypothesis that we will test here. We will elucidate the mechanisms of these fascinating enzymes to develop an integrated understanding of their complex oxidation chemistry.